During the last year, we continued define the molecular events that regulate hematopoietic stem cell (HSC) quiescence, survival, self-renewal and, myeloid cell lineage commitment and differentiation. We have focused our efforts on transcription factors since they are essential for stem cell growth and differentiation, and are frequently deregulated during the development of leukemias and lymphomas. We previously found that the helix loop helix (HLH) transcription factor, inhibitor of differentiation 1 (Id1), is induced during the early stages of myeloid development, and can instruct hematopoietic stem cells toward a myeloid versus erythroid and lymphoid cell fate. Furthermore, we discovered that deregulated expression of Id1 immortalizes hematopoietic progenitor cells in vitro, and leads a myeloproliferative disease (MPD) in vivo. In addition, we discovered that Id1 and Id2 genes are over expressed in human acute myelogenous leukemia (AML), and may represent a potential therapeutic target to treat human hematopoietic malignancies. In this regard, we found that reducing the levels of Id gene expression in human leukemic cell lines using Id1 siRNA inhibits cell growth in vitro. Future studies are planned to determine if we can inhibit or promote the differentiation of AML patient samplesin vitro, andin vivo when transplanted into severe combined immuno-deficient (SCID) mice. We have extended these studies to determine if Id1 is required for normal hematopoietic development using Id1-/- mice. Id-/- mice are viable and show no obvious defects. However, these mice have hematopoietic phenotypes including increased hematopoietic stem/progenitor cell cycling and defects in B cell and myeloid cell development. We determined that the hematopoietic phenotype observed in the Id1-/- mice was not intrinsic to the Id1-/- hematopoietic cells, but were due to defects in the microenvironment or niche. We found that stromal cells that lack Id1-/- expression can not support normal hematopoietic development in long term bone marrow cultures in vitro, suggesting that Id1 is required for the differentiation or function of the stromal cells. The Id1-/- stromal cells show altered hematopoietic growth factor (HGF) production including GM-CSF, SCF, G-CSF, SDF-1, which could explain, in part, the hematopoietic phenotype in Id1-/- mice. In addition, these mice showed a defect in the number of mesenchymal stem cells, which suggest that Id1 could also affect the development of specific stromal cell lineages including osteoblasts and adipocytes. Future experiments are planned to generate Id1 conditional knock out mice to specifically delete Id1 in endothelial cells, osteoblasts and other stromal cell populations. In addition, experiments are planned to determine if loss of Id1 gene expression in the microenvironment could contribute hematopoietic malignancy by promoting the survival and proliferation of tumor cells (tumor cell niche). Id proteins (Id1, Id2 and Id3) have been shown to be negative regulators of lymphoid development by antagonizing E protein function. However, it is not known if all Id proteins are physiologically required for lymphocyte development. By analyzing Id2-/- mice we discovered that Id2 is required for B cell development in vivo. In addition, we determined that Id2 positively regulates erythroid development by antagonizing the function of Pu.1. Thus, Id2 regulates lymphoid and erythroid development via the interaction with different transcription factor target proteins. Future studies are planned to evaluate the role of Id2 in regulating the survival, proliferation and self-renewal of HSC. In an effort to identify novel transcriptional regulators of myeloid cell growth and differentiation, we have compared the global gene expression profile of undifferentiated and differentiating hematopoietic progenitor cells. We have identified a novel zinc finger transcription factor of unknown function, POGZ, which is down regulated during the early stages of myeloid development. We have generated a mouse strain with a targeted deletion of POGZ. POGZ-/- mice do not survive beyond the first few hours of life, and die at birth of unknown causes, suggesting that POGZ is essential for mouse survival. We have discovered that fetal liver hematopoietic cell development is impaired in POGZ-/- mice, which includes a dramatic decrease in cellularity. In addition, fetal thymic development is arrested at a very early stage of development suggesting that POGZ is required for thymic development. Future studies are planned to transplant POGZ fetal liver cells into wild type recipient mice to evaluate the development of the entire hematopoietic system, and to determine if any of the observed defects are intrinsic to hematopoietic cells. Alternatively, POGZ will be deleted in adult hematopoietic cells using the POGZ conditional mice established in our lab, and mx1-cre and vav-cre transgenic mice.